The overall objective of the proposed research is to characterize the low density lipoprotein (LDL) receptor gene and to understand how it is altered in humans with familial hypercholesterolemia. Our initial goal will be the isolation of a complementary DNA clone to the bovine LDL receptor mRNA via the techniques of polyribosome immune precipitation and oligodeoxyribonucleotide hybridization with probes derived from sequence data of at least two regions of the bovine protein. We then propose to use this clone as a hybridization probe to identify human receptor cDNAs. Our second objective will be to characterize a full-length human cDNA clone at the primary structure level by standard DNA sequencing methodologies. From these results we will derive a series of oligodeoxyribonucleotides which span the entire cDNA and which are spaced approximately 200 base pairs apart. In future studies, this series of primers will be used to define the lesions present in mutant receptor genes. Our third objective is the isolation and characterization of the human LDL receptor gene from a bacteriophage Lambda library. We will use the cDNA clones as probes to identify the gene in these recombinants and will characterize it with respect to size, restriction endonuclease mapping, and its exon-intron arrangement. The results of the above studies will provide a framework for interpreting changes which are present in mutant receptor genes isolated from familial hypercholesterolemic patients. The final objective is to characterize two different mutant LDL receptor genes that occur in patients with familial hypercholesterolemia. These two mutations are of interest because they alter the size of the receptor protein and disrupt its synthesis, processing, and transport to the cell surface.